Oligonucleotides for RNA interference and biological applications thereof

Abstract

The invention relates to compositions comprising double-stranded oligonucleotides of identical or different sequences and/or length, said oligonucleotides having sequences 3′ N 1 N 2 . . . N i-1 N i . . . N j 5′ wherein— 3′ N i . . . N j 5′ is half of a double-stranded 19-28 mer oligonucleotide of sequence complementary to a target nucleic acid sequence present in a living cell, and— 3′ N 1 . . . N i-1 5′ is a 3-50 mer overhang of sequence allowing oligomerization of said double-stranded oligonucleotide. Compositions of transfection comprising said oligonucleotide compositions and there used for therapeutical application.

We claim:   
     
       1. A method for in vivo inhibition of gene expression comprising administering to a mammal composition for RNA interference comprising oligomerized double-stranded nucleic acids comprising a plurality of identical or different double-stranded nucleic acids and optionally a linker, wherein each of said identical or different double-stranded nucleic acids consists of a first distinct oligonucleotide and a second distinct oligonucleotide, wherein each of said first and second distinct oligonucleotides consists of 19-28 ribonucleotides at the 5′-end and 3-50 dT or dT and dA nucleotides at the 3′-end, wherein the 19-28 ribonulceotides at the 5′-end of the first distinct oligonucleotide are base-paired to the 19-28 ribonucleotides at the 5′-end of the second distinct oligonucleotide and wherein 3-50 dT or dT and dA nucleotides at the 3′-end of the first distinct oligonucleotide and at the 3′-end of the second distinct oligonucleotide are unpaired overhanging sticky dT or dT and dA nucleotides, such that the unpaired overhanging sticky dT or dT and dA nucelotides at the 3′-end of the first distinct oligonucleotide of one of the plurality of identical or different double-stranded nucleic acids are complementary to the unpaired overhanging sticky dT or dT and dA nucleotides at the 3′-end of the second distinct oligonucleotide of another of the plurality of identical or different double-stranded nucleic acids or to a linker having unpaired overhanging sticky dT or dT and dA nucleotides, and wherein said complementary unpaired overhanging sticky dT or dT and dA nucleotides of the plurality of identical or different double-stranded nucleic acids or of the linker are hybridized to form said oligomerized double-stranded nucleic acids. 
     
     
       2. A method for in vitro inhibition of gene expression comprising delivering to eukaryotic cells a composition for RNA interference comprising oligomerized double-stranded nucleic acids comprising a plurality of identical or different double-stranded nucleic acids and optionally a linker, wherein each of said identical or different double-stranded nucleic acids consists of a first distinct oligonucleotide and a second distinct oligonucleotide, wherein each of said first and second distinct oligonucleotides consists of 19-28 ribonucleotides at the 5′-end and 3-50 dT or dT and dA nucleotides at the 3′-end, wherein the 19-28 ribonulceotides at the 5′-end of the first distinct oligonucleotide are base-paired to the 19-28 ribonucleotides at the 5′-end of the second distinct oligonucleotide and wherein 3-50 dT or dT and dA nucleotides at the 3′-end of the first distinct oligonucleotide and at the 3′-end of the second distinct oligonucleotide are unpaired overhanging sticky dT or dT and dA nucleotides, such that the unpaired overhanging sticky dT or dT and dA nucelotides at the 3′-end of the first distinct oligonucleotide of one of the plurality of identical or different double-stranded nucleic acids are complementary to the unpaired overhanging sticky dT or dT and dA nucleotides at the 3′-end of the second distinct oligonucleotide of another of the plurality of identical or different double-stranded nucleic acids or to a linker having unpaired overhanging sticky dT or dT and dA nucleotides, and wherein said complementary unpaired overhanging sticky dT or dT and dA nucleotides of the plurality of identical or different double-stranded nucleic acids or of the linker are hybridized to form said oligomerized double-stranded nucleic acids. 
     
     
       3. The method according to  claim 1 , wherein said administering comprises introducing said composition for RNA interference into living cells and liberating said double stranded RNA in cells, which mediates sequence-specific RNA interference and mRNA degradation of one or many targeted genes in animals or humans. 
     
     
       4. The method according to  claim 2 , wherein said method mediates sequence-specific RNA interference and mRNA degradation of one or many targeted genes in eukaryotic cells in culture. 
     
     
       5. The method according to  claim 1 , wherein said mammal is in need of treatment of a cancer or a viral infection and said cancer or viral infection is treated by said administering. 
     
     
       6. The method according to  claim 5 , wherein said cancer is bladder cancer, prostate cancer or leukemia. 
     
     
       7. The method according to  claim 5 , wherein said viral infection is human immunodeficiency virus infection or hepatitis virus infection. 
     
     
       8. A method for in vivo inhibition of gene expression comprising administering to a mammal a composition for RNA interference comprising oligomerized double-stranded nucleic acids comprising a plurality of identical or different double-stranded nucleic acids and optionally a linker, wherein each of said identical or different double-stranded nucleic acids consists of a first distinct oligonucleotide and a second distinct oligonucleotide, wherein each of said first and second distinct oligonucleotides consists of 19-28 ribonucleotides at the 5′-end and 3-50 dT or dT and dA nucleotides at the 3′-end, wherein the 19-28 ribonulceotides at the 5′-end of the first distinct oligonucleotide are base-paired to the 19-28 ribonucleotides at the 5′-end of the second distinct oligonucleotide and wherein 3-50 dT or dT and dA nucleotides at the 3′-end of the first distinct oligonucleotide and at the 3′-end of the second distinct oligonucleotide are unpaired overhanging sticky dT or dT and dA nucleotides, such that the unpaired overhanging sticky dT or dT and dA nucleotides at the 3′-end of the first distinct oligonucleotide of one of the plurality of identical or different double-stranded nucleic acids are complementary to the unpaired overhanging sticky dT or dT and dA nucleotides at the 3′-end of the second distinct oligonucleotide of another of the plurality of identical or different double-stranded nucleic acids or to a linker having unpaired overhanging sticky dT or dT and dA nucleotides, and wherein said complementary unpaired overhanging sticky dT or dT and dA nucleotides of the plurality of identical or different double-stranded nucleic acids or of the linker are hybridized to form said oligomerized double-stranded nucleic acids and a transfection agent or formulation. 
     
     
       9. A method for in vitro inhibition of gene expression comprising delivering to eukaryotic cells a composition for RNA interference comprising oligomerized double-stranded nucleic acids comprising a plurality of identical or different double-stranded nucleic acids and optionally a linker, wherein each of said identical or different double-stranded nucleic acids consists of a first distinct oligonucleotide and a second distinct oligonucleotide, wherein each of said first and second distinct oligonucleotides consists of 19-28 ribonucleotides at the 5′-end and 3-50 dT or dT and dA nucleotides at the 3′-end, wherein the 19-28 ribonulceotides at the 5′-end of the first distinct oligonucleotide are base-paired to the 19-28 ribonucleotides at the 5′-end of the second distinct oligonucleotide and wherein 3-50 dT or dT and dA nucleotides at the 3′-end of the first distinct oligonucleotide and at the 3′-end of the second distinct oligonucleotide are unpaired overhanging sticky dT or dT and dA nucleotides, such that the unpaired overhanging sticky dT or dT and dA nucleotides at the 3′-end of the first distinct oligonucleotide of one of the plurality of identical or different double-stranded nucleic acids are complementary to the unpaired overhanging sticky dT or dT and dA nucleotides at the 3′-end of the second distinct oligonucleotide of another of the plurality of identical or different double-stranded nucleic acids or to a linker having unpaired overhanging sticky dT or dT and dA nucleotides, and wherein said complementary unpaired overhanging sticky dT or dT and dA nucleotides of the plurality of identical or different double-stranded nucleic acids or of the linker are hybridized to form said oligomerized double-stranded nucleic acids and a transfection agent or formulation. 
     
     
       10. The method according to  claim 8 , wherein said transfection agent is a linear polyethyleimine. 
     
     
       11. The method according to  claim 9 , wherein said transfection agent is a linear polyethyleimine. 
     
     
       12. The method according to  claim 8 , wherein said transfection formulation comprises at least a delivery reagent and other components stabilizing the formulation targeting tissues or organs of said mammal or increasing transfection efficiency. 
     
     
       13. The method according to  claim 9 , wherein said transfection formulation comprises at least a delivery reagent and other components stabilizing the formulation, targeting the cells or increasing transfection efficiency. 
     
     
       14. The method according to  claim 8 , wherein said transfection agent or formulation is not immunogenic. 
     
     
       15. The method according to  claim 1 , wherein said composition is delivered to cytoplasm.